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Individuals harboring germ-line DICER1 mutations are predisposed to a rare cancer syndrome, the DICER1 Syndrome or pleuropulmonary blastoma-familial tumor and dysplasia syndrome [online Mendelian inheritance in man (OMIM) #601200]. In addition, specific somatic mutations in the DICER1 RNase III catalytic domain have been identified in several DICER1-associated tumor types. Pituitary blastoma (PitB) was identified as a distinct entity in 2008, and is a very rare, potentially lethal early childhood tumor of the pituitary gland. Since the discovery by our team of an inherited mutation in DICER1 in a child with PitB in 2011, we have identified 12 additional PitB cases. We aimed to determine the contribution of germ-line and somatic DICER1 mutations to PitB. We hypothesized that PitB is a pathognomonic feature of a germ-line DICER1 mutation and that each PitB will harbor a second somatic mutation in DICER1. Lymphocyte or saliva DNA samples ascertained from ten infants with PitB were screened and nine were found to harbor a heterozygous germ-line DICER1 mutation. We identified additional DICER1 mutations in nine of ten tested PitB tumor samples, eight of which were confirmed to be somatic in origin. Seven of these mutations occurred within the RNase IIIb catalytic domain, a domain essential to the generation of 5p miRNAs from the 5' arm of miRNA-precursors. Germ-line DICER1 mutations are a major contributor to PitB. Second somatic DICER1 "hits" occurring within the RNase IIIb domain also appear to be critical in PitB pathogenesis.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is the most common undifferentiated ovarian malignancy in women under 40 years of age. We sequenced the exomes of six individuals from three families with SCCOHT. After discovering segregating deleterious germline mutations in SMARCA4 in all three families, we tested DNA from a fourth affected family, which also carried a segregating SMARCA4 germline mutation. All the familial tumors sequenced harbored either a somatic mutation or loss of the wild-type allele. Immunohistochemical analysis of these cases and additional familial and non-familial cases showed loss of SMARCA4 (BRG1) protein in 38 of 40 tumors overall. Sequencing of cases with available DNA identified at least one germline or somatic deleterious SMARCA4 mutation in 30 of 32 cases. Additionally, the SCCOHT cell line BIN-67 had biallelic deleterious mutations in SMARCA4. Our findings identify alterations in SMARCA4 as the major cause of SCCOHT, which could lead to improvements in genetic counseling and new treatment approaches.

DICER1 is an endoribonuclease responsible for the production of mature microRNAs which are small, single-stranded RNA molecules that regulate gene expression post-transcriptionally by binding to mRNA and repressing the expression of target genes. Germ-line mutations in DICER1 are responsible for a rare cancer syndrome, including tumors that can co-occur with multinodular goiter (MNG). Using Sanger sequencing, we screened all DICER1 exons and intron boundaries in 20 suspected mutation carriers: nine with ovarian sex cord-stromal tumors (including Sertoli-Leydig cell tumors (SLCTs)), five with pleuropulmonary blastoma, one with cystic nephroma, one with nasal chondromesenchymal hamartoma and four with more than one manifestation suggestive of a germ-line DICER1 mutation. All were negative for any apparently deleterious variants. We developed a Multiplex Ligation-based Probe Amplification assay for DICER1 to screen for large deletions or duplications. Synthetic oligonucleotides were designed to cover all exons in three probe-mixes. In a child with a SLCT and MNG, and in her mother and brother (both diagnosed with MNG), we identified a heterozygous germ-line deletion of approximately 3 kilobases that eliminates exon 21 of DICER1 and two-thirds of intron 21, accompanied by an insertion of a G nucleotide at the 3' end of the deletion (c.3270-6_4051-1280delinsG). This allele is expressed in the patient's cDNA, creating an out-of-frame deletion predicted to result in a truncated protein (r.3270_4050del; p.Tyr1091Ser*28). Our novel finding of a disease-causing large deletion in DICER1 emphasizes the need to include assays that can detect rearrangements, duplications and deletions in any DICER1 screening protocol.

Organogels can be prepared by immobilizing an organic phase into a threedimensional network coming from the self-assembly of a low molecular weight gelator molecule. In this work, an injectable subcutaneous organogel system based on safflower oil and a modified-tyrosine organogelator was evaluated in vivo for the delivery of rivastigmine, an acetylcholinesterase (AChE) inhibitor used in the treatment of Alzheimer’s disease. Different implant formulations were injected and the plasmatic drug concentration was assayed for up to 35 days. In parallel, the inhibition of AChE in different brain sections and the biocompatibility of the implants were monitored. The pharmacokinetic profiles were found to be influenced by the gel composition, injected dose and volume of the implant. The sustained delivery of rivastigmine was accompanied by a significant prolonged inhibition of AChE in the hippocampus, a brain structure involved in memory. The implant induced only a minimal to mild chronic inflammation and fibrosis, which was comparable to poly(D,L-lactide-co-glycolide) in situ-forming implants. These findings suggest that tyrosine-based organogels could represent an alternative approach to current formulations for the sustained delivery of cholinesterase inhibitors., Faculté de Pharmacie

Small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is an aggressive tumor and the most common type of undifferentiated ovarian malignancy presenting at less than 40 years of age. Its cause and histogenesis remain unknown. We sequenced the exomes of individuals from three familial cases of SCCOHT. Subsequently, we used whole exome sequencing, Sanger sequencing and immunohistochemistry to analyze germline and tumor DNA from three additional familial cases, 35 non-familial cases and one SCCOHT cell line (BIN-67). DNA sequencing revealed likely deleterious germ-line mutations in the chromatin remodeling gene SMARCA4 in all four familial cases of SCCOHT where DNA was available. This was accompanied by either a somatic mutation or loss of the wild-type allele in the tumor. BIN-67 contained bi-allelic deleterious mutations in SMARCA4. Sequencing of 24 non-familial pathologically-confirmed SCCOHT cases revealed at least one germ-line or somatic likely deleterious SMARCA4 mutation in 22 cases. Immunohistochemical (IHC) analyses of these and an additional 11 tumors showed loss of BRG1 protein in 38/40 cases. Our findings identify alterations in SMARCA4 as a major cause of SCCOHT, which could pave the way for genetic counseling and new treatment approaches. Citation Format: Leora Witkowski, Jian Carrot-Zhang, Steffen Albrecht, Nancy Hamel, Eva Tomiak, David Grynspan, Emmanouil Saloustros, Catherine Gilpin, Rachel Silva-Smith, François Plourde, Barbara Rivera, Ester Castellsagué, Mona Wu, Somayyeh Fahiminiya, Javad Nadaf, Avi Saskin, Madeleine Arseneault, Rouzan G. Karabakhtsian, Elizabeth A. Reilly, Frederick R. Ueland, Anna Margiolaki, Kitty Pavlakis, Sharon M. Castellino, Janez Lamovec, Lawrence M. Roth, Thomas M. Ulbright, Tracey Bender, Michel Longy, Andrew Berchuck, Marc Tischkowitz, Reiner Siebert, Inga Nagel, Vassilis Georgoulias, Colin J.r. Stewart, Glenn McCluggage, Jocelyne Arseneau, Blaise A. Clarke, Yasser Riazalhosseini, Martin Hasselblatt, Jacek Majewski, William D. Foulkes. Germ-line and somatic SMARCA4 mutations characterize small cell carcinoma of the ovary, hypercalcemic type. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-89. doi:10.1158/1538-7445.AM2014-LB-89